1. Field of the Invention
This invention relates to immunometric assays for simultaneously detecting and/or measuring the amount of at least two antigens in samples such as serum, urine or other body fluids. This invention is also directed to test kits for conducting such immunomeric assays.
2. Description of Related Disclosures
Immunoassay techniques are increasingly being used for diagnostic purposes to measure the concentration of antigens, particularly those which are physiologically harmful, in various types of body fluids such as serum and urine. Immunoassay methods, which include competitive immunoassays as well as immunometric assays, rely on interaction between the antigen in question and one or more antibodies to form a complex which is detectable by means of a label on the antigen or antibody.
When the antigens have more than one type of binding site they are advantageously detected by immunometric assays. In this method, a soluble antibody labeled with a signal moiety is employed in conjunction with an unlabeled antibody bound to a solid carrier that is insoluble in the fluid being tested. These two types of antibodies form a ternary complex with the antigen which is detected by the label of the soluble antibody. This so-called "sandwich" assay, where the antigen binds two antibodies to its surface at different locations, is described generally by Wide in Radioimmunoassay Methods, ed. by Kirkham et al. (Edinburgh: E. & S. Livingstone, 1970) at pages 199-206. U.S. Pat. No. 4,343,896 also describes use of an immunometric assay for detecting an at least bivalent antigen in a liquid sample.
U.S. Pat. No. 4,376,110 describes a technique using a one-to-one sandwich immunometric assay where one monoclonal antibody is presented in a soluble labeled form and the second monoclonal antibody is bound to an insoluble carrier. This technique represents advantages over prior art techniques using polyclonal antibodies in the sandwich assay. For example, the simultaneous and reverse assays using monoclonal antibodies are more sensitive and rapid that the assays using polyclonal antibodies. In addition, the formation of the soluble sandwich complex does not compete with formation of the desired insoluble complex. PCT Patent Publication No. WO 82/02661 teaches a similar assay. European Patent Application No. 0,045,103 published Feb. 3, 1982 describes an immunochemical determination method where at least two types of monoclonal antibodies are used directed against the same antigen to be determined. U.S. Ser. No. 466,798 filed Feb. 16, 1983 to Murad entitled "Two-site Immunoassays Using Monoclonal Antibodies of Different Classes or Subclasses and Test Kits for Performing Same" discloses a further aspect of the sandwich immunometric assay where the two antibodies employed to detect the antigen are of different immunoglobulin classes or subclasses. U.K. Pat. No. 2,074,727, German Patent Publication No. 3205849, and European Patent Publication Nos. 48,357 and 44,219 describe similar such assays.
It is often desirable, however, to detect more than one antigen in a fluid simultaneously due to small sample volume, low reagent cost and short overall assay time. M. Kuriyama et al., JNCI, 68, 99-105 (1982) describe the advantage of using a combination test of tissue-specific markers to detect the presence of two proteins of human prostate-specific origin, namely, prostatic acid phosphatase and prostate antigen. Kuriyama et al., however, did not detect the two antigens by a simultaneous method but rather combined the results of the separate measurements of each antigen.
Mitsuma et al., Biochem. Biophys. Res. Commun., 46, 2107-2113 (1972), Ljunggren et al., Acta Endocrinol., 81, 487-494 (1976) and Haynes et al., Ann. Clin. Biochem., 14, 12-15 (1977) used indirect competitive radioimmunoassay to measure more than one component in a single tube by labeling antigens with two different iodine isotopes, including .sup.131 I which has a half life of only 8 days. Vihko et al., Clin. Chem., 27, 1744-1746 (1981) disclose immobilizing two types of antibodies on different sections of test tubes and joining them to produce a single multicomponent tube. The haptens are labeled with .sup.125 I, which is detected by indirect radioimmunoassay. This method represents a relatively complex procedure involving separation of the solid phase carrier.
U.S. Pat. No. 4,315,907 and J. Immunol. Methods, 26, 381 (1979) describe a specific binding assay to determine multiple antigens employing solid-phase binding agents corresponding to each antigen which are differentially separable, as well as labeled binding agents for each antigen. Each solid-phase bound species is separated from all the other species after incubation.
C. Blake et al., Clinical Chemistry, 28, 1469-1473 (1982) describe the simultaneous enzyme immunoassay of two thyroid hormones which has advantages over radioisotopes in that it is stable and allows for facile differential measurement. In this assay a mixture of two conjugates labeled with two different enzymes is used to detect both antigens simultaneously in a fluid. The enzymes form products easily distinguishable from each other by absorption spectrophotometry. Blake et al. used two assay compounds, i.e., two antibodies immobilized on two separate solid carriers, to detect both antigens, rather than a single assay compound.
Prostate antigen (PA) and prostatic acid phosphatase (PAP) are two distinct antigenic proteins of human prostate-specific origin. Biochemically, PA is a glycoprotein of molecular weight 33,000 with a pI of 6.9, whereas PAP has a molecular weight of 100,000 and a multiple pI ranging from 4.2 to 5.5. Antisera specifically react toward each antigen and to not cross-react with each other. Although not specific to prostate tumors, PAP and PA in combination are promising markers in detecting prostate cancer because they may reflect different aspects of neoplastic transformation in prostate cancer. Thus, detecting both markers simultaneously represents a diagnostic tool of considerable importance.